Fluorescence Imaging of Single-Copy DNA Sequences within the Human Genome Using PNA-Directed Padlock Probe Assembly

We present an approach for fluorescent in situ detection of short, single-copy sequences within genomic DNA in human cells. The single-copy sensitivity and single-base specificity of our method is achieved due to the combination of three components. First, a peptide nucleic acid (PNA) probe locally opens a chosen target site, which allows a padlock DNA probe to access the site and become ligated. Second, rolling circle amplification (RCA) generates thousands of single-stranded copies of the target sequence. Finally, fluorescent in situ hybridization (FISH) is used to visualize the amplified DNA. We validate this technique by successfully detecting six single-copy target sites on human mitochondrial and autosomal DNA. We also demonstrate the high selectivity of this method by detecting X- and Y-specific sequences on human sex chromosomes and by simultaneously detecting three sequence-specific target sites. Finally, we discriminate two target sites that differ by 2 nt. The PNA-RCA-FISH approach is a distinctive in situ hybridization method capable of multitarget visualization within human chromosomes and nuclei that does not require DNA denaturation and is extremely sequence specific. [Display omitted] ► PNA-directed assembly of the labeling complex on duplex DNA ► Padlock probes combined with rolling circle amplification for signal amplification ► Multitarget visualization within the human genome with high sequence selectivity Yaroslavsky and Smolina report a method for fluorescent in situ detection of short, single-copy sequences within human genomic DNA. The method combines the use of PNA probes to locally open dsDNA with rolling circle amplification to generate thousands of copies of the target sequence visualized by fluorescence.